Retroviruses infect a wide variety of cells and are ideal tools for the delivery of genes to target cells. They are furthermore an ideal tool to stably integrate a heterologous sequence in the genome of a target cell, since the infecting retrovirus is able to integrate the DNA form of its RNA genome into the genome of the target cell. Thus, all daughter cells of a retroviral infected cell carry the retroviral vector DNA possibly comprising a heterologous gene.
A retroviral genome consists of a RNA molecule with the structure R-U5-gag-pol-env-U3-R. For the development of a retroviral vector (RV) said retroviral genome can be modified by replacing the genes gag-pol-env-encoding viral proteinsxe2x80x94with one or more genes of interest such as marker genes or therapeutic genes. To generate a recombinant retroviral particle and a packaged RV, respectively, the principle of a retroviral vector system is used. This system consists of two components: the RV itself in which the genes encoding the viral proteins have been replaced, and a packaging cell which provides the modified RV with the missing viral proteins. This packaging cell has been transfected with one or more plasmids carrying the genes enabling the modified RV to be packaged, but lacks the ability to produce replication competent viruses.
After introduction of the vector into the packaging cell line, the RV is transcribed into RNA. This RNA which represents the recombinant retroviral genome is packaged by the viral proteins produced by the packaging cell to form retroviral particles which bud from the packaging cell. These particles are further used to infect a target cell. In the target cell the RNA genome is released again from the particle, reverse transcribed and stably integrated into the cellular genome.
Therefore, RVs are currently the method of choice for a stable transfer of therapeutic genes into a target cell in a variety of approved protocols both in the USA and in Europe. However, most of the protocols require that the infecting of target cells with the RV carrying the therapeutic gene occurs in vitro. Subsequently, successful infected cells are returned to the affected individual. Advantageously, such ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use. Furthermore, the ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated.
Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated cultured and then reintroduced to a patient. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the RV, or cells producing the RV, are directly administered to the patient in the form of an injection or simple implantation of RV producing cells.
This kind of in vivo approach, of course, introduces a variety of new problems. First of all safety considerations have to be addressed. One serious safety risk is that virus will be produced, possibly form an implantation of virus producing cells. Thus, there will be no opportunity to precheck said produced virus. Another problem is that the proviral form of the retroviral genome integrates randomly in the genome of infected cells. This random integration can result in an integration directly into a cellular gene or into the vicinity of a cellular gene, leading to new genomic arrangements. As a result of this function of the cellular gene can be altered or lost. In the case that the cellular gene is involved in the regulation of growth control, uncontrolled proliferation of the cell may result. Therefore, using RV in gene therapeutic applications there is a potential risk that simultaneously to the repair of one genetic defect with retroviral vectors, a second defect can be established resulting in uncontrolled proliferation, and thus, in tumor development.
It is therefore an object of the invention to provide a safe retroviral vector which prevents random integration of the recombinant viral genome into genes or into the vicinity of genes of a target cell genome, thus, preventing genomic rearrangements of the target cell genome.
The invention inter alia comprises the following, alone or in combination:
A retroviral vector comprising one or more heterologous nucleic acid sequence(s) as well as at least one sequence allowing site-specific integration of said heterologous sequence(s) into a non-coding region of a genome;
the retroviral vector as above, wherein the sequence(s) allowing site specific integration is inserted at the U3 region(s) and/or the U5 region(s) of the retroviral Long Terminal Repeat (LTR);
the retroviral vector as above, wherein the sequence allowing site specific integration is an Inverted Terminal Repeat (ITR) sequence of Adeno-associated virus (AAV); the retroviral vector as any above, wherein the genome is a chromosome of a mammal, including human;
the retroviral vector as above, wherein the chromosome is chromosome 19;
the retroviral vector as any above, wherein at least one of the heterologous nucleic acid sequence(s) is a heterologous gene relevant for the treatment of a viral infection or the treatment of a genetic, metabolic, proliferative or any other relevant disorder or disease;
the retroviral vector as any above, wherein at least one of the heterologous nucleic acid sequence(s) is a sequence encoding an integration-mediating protein;
the retroviral vector as above, wherein the integration-mediating protein is the AAV Rep protein;
the retroviral vector as above, wherein the sequence encoding for the integration-mediating protein is under transcriptional control of an inducible promoter;
a retroviral vector system comprising the vector as any above as a first component, and a packaging cell harboring at least one DNA construct encoding for proteins required for said vector to be packaged;
the retroviral vector system as above, wherein the packaging cell synthesizes a mutated or a completely or partially deleted retroviral integrase (IN);
a retroviral particle comprising a retroviral vector as any above;
the retroviral particle as above obtainable by transfecting a packaging cell of a retroviral vector system as above with the retroviral vector as above;
a retroviral provirus produced by infection of target cells with the retroviral particle as above;
mRNA of a retroviral provirus as above;
RNA of the retroviral vector as any above;
cDNA of the RNA as above;
a host cell infected with the retroviral particles as above;
a method for introducing homologous and/or heterologous nucleotide sequences into target cells comprising infecting the target cells with retroviral particles as above;
the retroviral vector as any above and/or the retroviral particle as above and/or the retroviral vector system as above for the use in the treatment of a viral infection or the treatment of a genetic, metabolic, proliferative or any other relevant disorder or disease;
use of the retroviral vector as any above and/or the retroviral particle as above and/or the retroviral vector system as above for producing a pharmaceutical composition for the treatment of a viral infection or the treatment of a genetic, metabolic, proliferative or any other relevant disorder or disease;
a pharmaceutical composition containing a therapeutically effective amount of the retroviral vector as any above and/or the retroviral particle as above and/or the retroviral vector system as above;
a method of treating a viral infection or a genetic, metabolic, proliferative or any other relevant disorder or disease comprising administering to a subject in need thereof a therapeutically effective amount of the retroviral particle as above and/or the retroviral vector system as above.
The basic idea underlying the present invention is the provision of a recombinant retroviral vector which specifically integrates into a targeted region of a target cell genome. Thus, to achieve the foregoing and other objects, the present invention provides a retroviral vector (RV) comprising at least one integration-mediating sequence, wherein said sequence is site-specific for a targeted region of a target cell genome. The sequence is, thus, included into the recombinant retroviral genome, preferably within the retrovirus-derived sequences, and is transferred into a target cell. For transfer, the retroviral vector is preferably packaged into a retroviral particle. After transfer into the target cell the retroviral vector is integrated into a specific site of the target cell genome, whereby said specific site is determined by the site specific integration-mediating sequence included in the vector.
The term xe2x80x9csite specificxe2x80x9d integration-mediating sequence includes that the sequence is of non-retroviral origin. Integration-mediating sequences of retroviral origin are generally non-site-specific and do, thus, only allow random integration into the genome of the target cell. The inventors of the present invention showed for the first time that retroviral sequences can be integrated into a target cell by non-retroviral sequences. Integration-mediating sequences, specifically those of non-retroviral origin, are known to form stable secondary structures, as, e.g., xe2x80x9chairpin loopxe2x80x9d structures, which are generally inaccessible for enzymes. Such structures may, thus, inhibit enzyme activities. When now considering the life cycle of a retrovirus, it was not awaited that a non-retroviral integration-mediating sequence would mediate integration of the retroviral genome into the target cell genome:
When a retroviral genome has entered a target cell, the retroviral RNA is reverse transcribed into DNA and, subsequently, the DNA is integrated into the host cell genome. The integrated DNA is further transcribed into mRNA, wherein transcription starts at the U5-region of the 5xe2x80x2-LTR and ends at the U3-region of the 3xe2x80x2-LTR. When the integration-mediating sequence is included in the retroviral vector, the integration-mediating sequence must also be reverse-transcribed, must be integrated into the target cell genome and, subsequently, must be transcribed into the mRNA. However, reverse transcription, integration and transcription of the DNA into RNA are all dependent on catalytic functions of specific enzymes. Said catalytic functions can only develop after binding of the enzymes to the nucleic acid sequence. As mentioned above, non-retroviral integration-mediating sequences form secondary structures, which may hinder enzymes to bind to the nucleotide sequence. Accordingly, the skilled practitioner would have at first expected that the insertion of integration-mediating sequences into a retroviral vector would result in the formation of secondary structures, preventing binding of the enzyme catalyzing reverse transcription and thereby inhibiting reverse transcription. Even if it was awaited that reverse transcription would take place, the inhibition of the integration would have been further expected also due to the secondary structures of the integration-mediating sequences. However, without reverse transcription and integration of the retroviral vector the retroviral sequence is not stable. Hence, it was expected that the retroviral vector including a non-retroviral integration-mediating sequence would be lost shortly after it enters the target cell.
However, only assuming, it could be awaited that the retroviral vector would integrate into the target cell genome, the skilled practitioner would further not have expected that the integrated sequence would be transcribed and translated. More likely, the skilled practitioner would have expected that the large transcription complex necessary for transcription of the integrated DNA could not bind to the nucleic acid sequence again due to the secondary structures of the non-retroviral sequence and additionally due to the large size of the enzyme complex. Consequently, no synthesis of mRNA of the retroviral part included in the host cell genome was expected. Furthermore, since transcription of said mRNA starts at the retroviral promoter in the 5xe2x80x2-LTR, the mRNA also comprises the integration-mediating sequence. Hence, the mRNA was expected to again form secondary structures. Accordingly, it was further expected that the large translation complex could not anneal to the mRNA, resulting in no translation of the mRNA into protein. Accordingly, even if integration of the retroviral vector into the target cell genome was expected, the skilled practitioner would not have expected that protein encoded by sequences integrated into the retroviral vector would be produced. However, in contrast to all the above expectations, it was found that the retroviral vector according to the present invention is not only reverse transcribed and integrated, but that also proteins are produced from the sequence inserted into the vector.
Preferably, the integration-mediating sequence included in the retroviral vector is specific for a non-coding region of the target cell genome, i.e., due to the sequence allowing site-specific integration the RV interacts with a genomic region which does not contain any coding or regulatory sequences. Interaction and subsequent integration may occur by homologous recombination or to another, e.g. protein mediated, integration mechanism. Generally, the retroviral integration process is mediated by an integration-mediating enzyme, which is comprised in an infectious retroviral particle. The integration-mediating protein interacts with the sequence allowing site-specific integration encoded by the RV as well as with the site of integration within the region of the genomic sequence of the target cell. Thus, said target cell is infected by a retroviral particle comprising the RV and optionally an integration-mediating protein. Consequently, site-specific integration of the RV into a genomic region of a target cell occurs.
As a result of site-specific integration of the RV the risk of new genomic arrangements, e.g. leading to disregulations of gene products or uncontrolled cellular proliferation, is avoided. Thus, the RV according to the present invention is highly adapted for future in vivo, but also in vitro transfer of heterologous nucleic acid sequences to target cells of mammals, including humans. Thus, according to a further preferred embodiment of the present invention the vector additionally includes one or more heterologous nucleic acid sequence(s).
The term xe2x80x9cheterologousxe2x80x9d is used for any combination of DNA sequences that is not normally found intimately associated in nature. Accordingly, at least one of the heterologous nucleic acid sequences of RV as described above is a heterologous gene relevant for the treatment of a viral infection, a genetic, a metabolic, a proliferative or any other relevant disorder or disease. Therefore, heterologous genes which can be transferred to target cells by the RV according to the present invention are preferably, but not limited to one or more elements of the group consisting of marker genes, therapeutic genes, antiviral genes, antitumor genes, cytokine genes and/or toxin genes. The marker and therapeutic genes are preferably selected from genes such as xcex2-galactosidase gene, neomycin gene, Herpes Simplex Virus thymidine kinase gene, puromycin gene, cytosine deaminase gene, hygromycin gene, secreted alkaline phosphatase gene, guanine phosphoribosyl transferase (gpt) gene, alcohol dehydrogenase gene, hypoxanthine phosphoribosyl transferase (HPRT) gene, green fluorescent protein (gfp) gene, cytochrome P450 gene and/or toxin genes such as a subunit of diphtheria, pertussis toxin, tetanus toxoid.
To ensure that during the integration event the heterologous sequence(s) encoded by the RV integrates into a genomic non-coding region, said heterologous sequence(s) is flanked by one or more sequences allowing site-specific integration. Generally, it is possible to introduce the process of integration with a single copy of the sequence allowing site-specific integration, which in this case flanks only one end of the heterologous sequence to be integrated. However, in a preferred embodiment the sequences allowing site-specific integration flankxe2x80x94directly or at some distancexe2x80x94both sites of the heterologous sequences to be integrated. Thus, said sequences allowing site-specific integration are preferably inserted into the U3 region(s) and/or U5 region(s) of the retroviral LTR. Alternatively, said sequences allowing site-specific integration could be inserted joining the heterologous sequence to be integrated. In this case, only the heterologous sequence to be integrated without any further retroviral sequences will be site-specifically integrated. Therefore, in this case the RV serves only as a vehicle for the transport of the heterologous sequences to be integrated into the target cell.
The RV according to the present invention is particularly useful for the site specific integration into a non-coding region of a mammalian, including a human chromosome, since it is known that more than 90% of the mammalian genome consist of non-coding regions. In a further embodiment of the present invention the RV integrates specifically in a non-coding region, which is located on human chromosome 19. Said specific non-coding DNA region on human chromosome 19 was first described as the target site for the integration of Adeno-associated virus (AAV). For an integration into said non-coding region on chromosome 19, in still a further embodiment of the present invention, the sequences allowing site-specific integration of the RV are the so called Inverted Terminal Repeats (ITRs) of the AAV.
When combining these features of phylogenetic different viruses it was found as particularly advantageous that the resulting RV according to the present invention, can still accommodate a capacity of about 8 kb of heterologous DNA sequences, which can be targeted to a non-coding region in the genome. In comparison, all existing AAV based vectors can accommodate a maximum of about 4,5 kb of heterologous DNA in the presence of all coding region required for targeted integration into chromosome 19 (Dong et al., 1996, xe2x80x9cQuantitative analysis of the packaging capacity of recombinant adeno-associated virus,xe2x80x9d Hum. Gene Ther., 7(17): 2101-2112)). Unfortunately, this is too little to be of practical use for most gene therapies.
The present invention also provides a method for introducing a homologous or heterologous nucleic acid sequence into the genome of a target cell. According to this method, said sequence is included into a retroviral vector and transferred by this vector into the target cellxe2x80x94by transfection and/or infection with a retroviral particle including said vector. However, integration of said sequence into the target cell genome is catalyzed by a non-retroviral integration-mediating protein. It was surprisingly found that a non-retroviral integration-mediating protein can indeed mediate integration of sequences included in a retroviral vector. At the time the invention was made integration of vectors derived from, e.g., DNA viruses was only mediated by proteins also derived from a DNA virus, i.e. it was only common practice that for integration of vectors from a DNA organism the integration-mediating protein must also be derived from the same origin, namely from a DNA organism. Accordingly, for integration of a retroviral vector derived from an RNA genome and a retrovirus, respectively, only retroviral integrase was used. However, it was not expected that sequences included in a retroviral vector can be integrated by a non-retroviral integration-mediating protein.
In a preferred embodiment of the present invention the AAV-Rep protein is used for the site-specific integration of the RV. It was surprisingly found that the AAV integration-mediating Rep Protein can be used for targeted integration of the RV into the same non coding region of the chromosome 19 which this protein normally uses for the AAV integration process. As already indicated above, this was particularly unexpected, since a RV is based on a virus with RNA genome, while AAV belongs to the viruses with a DNA genome. According to these differences in genome structure also the regulation or integration mechanism is completely different. Whereas the integration of the retroviral genome is normally dependent on the enzyme integrase (IN), the site-specific integration of the AAV genome is mediated by the Rep protein. Since this protein is AAV-specific it was not expected that the integration of a foreign genome would be mediated by this protein. Additionally, it was not expected that a protein of a DNA virusxe2x80x94belonging to a completely different phylogenetic group when combined with a RNA virusxe2x80x94would mediate integration of a retroviral genome.
To provide a target cell with an integration-mediating protein, e.g. said AAV-Rep protein, one alternative is to directly incorporate the nucleic acid sequence encoding said protein in the RV. After infection of a target cell with the RV the integration-mediating protein, e.g. the AAV Rep protein, is directly synthesized in the target cell. Subsequently, the AAV Rep protein mediates site-specific integration of the RV.
Alternatively, the packaging cell provides the retroviral particle (RVP) with the integration-mediating protein, e.g. AAV Rep protein. In this case the integration-mediating protein is synthesized from the packaging cell and packaged into newly generated infectious retroviral particles (RVP). Subsequently, these particles are used to infect a target cell, and thereby, transfer said additional integration-mediating protein together with the RV into the target cell.
It is known that the expression of an integration-mediating protein, particularly of the AAV Rep protein, induces at higher concentrations toxic effects in cells. Accordingly, in a further embodiment of the present invention the expression of the integration-mediating protein as well as of the AAV Rep protein is under the transcriptional control of an inducible and/or a very weak promoter. The inducible promoters and/or very weak promoters are selected preferably, but not limited, from one or more elements of the group consisting of promoters inducible by Tetracycline, promoters inducible by HIV Tat transactivator, promoters inducible by glucocorticoid hormones, such as the MMTV promoters or promoters inducible by X-ray.
For the generation of RVP in a further embodiment of the invention a retroviral vector system is provided, which comprises the RV as described above as a first component and a packaging cell providing the proteins required for the RV to be packaged. The packaging cell line is selected preferable but not limited, from an element of the group consisting of psi-2, psi-Crypt, psi-AM, GP+E-86, PA317, GP+envAM-12, Fly A13, BOSC 23, BING, Fly RD 18, ProPak-X, -A.52 and -A.6, or of any of these supertransfected with recombinant constructs allowing expression of surface proteins from other enveloped viruses.
To ensure a high efficacy of site-specific integration of the RV the packaging cell according to a further embodiment of the present invention provides a Gag/Pol expression plasmid that does not encode a functional retroviral integrase (IN). Accordingly, the packaging cell is constructed in such a way that no functional retroviral IN which is encoded by the pol-region can be synthesized. For this, the packaging cell is generated using a DNA construct encoding a retroviral pol-region which incorporates mutations and/or partial or complete deletions of the pol-region. To introduce mutations or deletions in the pol-region leading to a non-functional IN preferably recombinant PCR technology is used.
The invention further provides retroviral particles comprising the RV of the invention as described above. These particles can be obtained by transfecting according to standard protocols the packaging cell as described above with RV as described above.
The invention includes a retroviral provirus, mRNA of a retroviral provirus according to the invention, any RNA resulting from a retroviral vector according to the invention and cDNA thereof, as well as target cells infected with a retroviral particle according to the invention.
A further embodiment of the invention provides a method for introducing homologous and/or heterologous nucleotide sequences into target cells comprising infecting a target cell population in vivo and in vitro with recombinant retroviral particles as described above. Furthermore, the retroviral vector, the retroviral particle, the retroviral vector system and the retroviral provirus as well as RNA thereof is used in the treatment of a viral infection or the treatment of a genetic, metabolic, proliferative or any other relevant disorder or disease.
The retroviral vector, the retroviral particle, the retroviral vector system and the retroviral provirus as well as RNA thereof is used for producing a pharmaceutical composition for in vivo and in vitro gene therapy in mammals including humans.
The invention further includes a method of treating a viral infection or a genetic, metabolic, proliferative or any other relevant disorder or disease comprising administering to a person in need thereof a therapeutically effective amount of the retroviral particle and/or the retroviral vector system and/or a pharmaceutical composition containing a therapeutically effective amount of the retroviral vector, vector system or particle.